1. Field of the Invention
The present invention relates to a method of plant transformation in which a foreign gene is introduced into the zygote in an isolated embryo sac and a transgenic plant is recovered. The present invention also relates to a plant-transformation approach whereby a foreign gene is introduced into an egg cell in an isolated embryo sac, the egg cell is fertilized with a sperm cell, and a transgenic plant is recovered. In addition, the present invention relates to plant transformation by introducing a foreign gene into isolated sperm cells, after which a transformed sperm cell, or the nucleus isolated from the transformed sperm cell, is fused with an egg in an isolated embryo sac and a transgenic plant is recovered. These methodologies yield uniformly transformed plants capable of transmitting a foreign gene to progeny.
2. Background
Numerous methods for plant transformation have been developed, including biological and physical plant transformation protocols. See, for example, Miki et al., "Procedures for Introducing Foreign DNA into Plants" in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 67-88 (CRC Press, 1993). In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and recovery of plants are available. See, for example, Gruber et al., "Vectors for Plant Transformation," loc. cit. at 89-119.
Production of transgenic plants first became routine through the use of Agrobacterium. The host range for Agrobacterium-mediated transformation is broad and includes not only dicotyledonous but also monocotyledonous plants such as rice and maize. Hiel et al., The Plant Journal 6: 271-282 (1994) and Ishida et al., Nature Biotechnol. 14: 745-750 (1996). Several methods of plant transformation, collectively referred to as "direct gene transfer," have been developed as an alternative to Agrobacterium-mediated transformation.
A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 .mu.m. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5: 27 (1987); Sanford, Trends Biotechnol. 6: 299 (1988); Sanford, Physiol. Plant 79: 206 (1990); Klein et al., Bio/Technology 10: 268 (1992).
Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J. 4: 2731 (1985); Christou et al., Proc Nat'l Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake of DNA into protoplasts using CaCl.sub.2 precipitation, polyvinyl alcohol, poly-L-ornithine or electroporation have also been reported. Hain et al., Mol. Gen. Genet. 199: 161 (1985); Draper et al., Plant Cell Physiol. 23: 451 (1982); Fromm et al., Nature 319; 791 (1986). Electroporation of protoplasts and plant cells in intact tissue is well documented. Luehrsen et al., in THE MAIZE HANDBOOK 613-615, Freeling et al. (eds.) (1994); deHalluin et al., Plant Cell 4: 1495-1505 (1992); Laursen et al., Plant Mol. Biol. 24: 51-61 (1994).
Finally, microinjection of DNA has been studied in a variety of plant cells including, for example, isolated protoplasts, callus, microspore-derived embryonic cells and apical meristem. Crossway et al., Mol. Gen. Genet. 202: 179-185 (1986); Toyoda et al., Plant Cell Rep. 7: 293-296 (1988); Neuhaus et al. Theor. Appl. Genet. 75: 30-36 (1987); Simmonds et al., Physiol. Plantarum 85: 197-206 (1992).
Although the production of gametes and zygotes by plants is well-understood, reproducible methods for in vitro manipulation and transformation of these cells are needed. In most plant species, embryo sacs are deeply enclosed in the sporophytic tissues and therefore the embryo sacs are difficult to manipulate. Microsporocytes and developing pollen grains are also embedded in sporophytic tissues.
Within the immature anther are cavities containing microsporocytes or pollen mother cells. Each mother cell undergoes two successive nuclear divisions to form a tetrad of four microspores. Each of these microspores may develop into a pollen grain. A microspore develops into a pollen grain by a thickening of the spore wall and a division of the microspore nucleus to form a vegetative cell and a generative cell.
Pollination involves the transfer of pollen from anther to stigma. Pollen germinates on the stigma and a pollen tube grows through the style and enters the tip of the ovule through the micropyle. Two male gametes or sperm cells are formed by division of the generative cell of the pollen grain. The sperm cells move through the pollen tube and are emptied into the embryo sac.
Within each ovule is a megasporocyte that undergoes two successive nuclear divisions to produce four megaspores. Three of the megaspores usually disintegrate while the fourth continues to undergo nuclear divisions and forms an eight nucleate embryo sac. The egg and two synergids are found near the micropyle while three nuclei are found on the opposite end of the embryo sac. Two polar nuclei remain in the center of the embryo. One sperm cell fuses with the egg cell to form the zygote which eventually develops into the embryo and a new plant. The second sperm fuses with the two polar nuclei to form the primary endosperm nucleus which divides many times to form the endosperm.
Methods for the isolation of living embryo sacs have been developed for plant species by using either micromanipulation or enzymatic digestion. Allington, "Micromanipulation of the Unfixed Cereal Embryo Sac," in THE EXPERIMENTAL MANIPULATION OF OVULE TISSUES 39-51 (Longman, N.Y., 1985); Theunis et al., Sex Plant Reprod. 4: 145-154 (1991); Wu et al., loc. cit. 6: 171-175 (1993). In vitro manipulation of fertilized embryo sacs frequently results in low viability or production of abnormal embryos. Leduc et al., loc. cit. 8: 313-317 (1995).
Many plant transformation systems, especially for cereals, involve callus-based selection protocols and extensive in vitro culturing. These methodologies are time consuming and increase the likelihood that somaclonal variants will arise that exhibit undesirable agronomic characteristics. Use of developmentally organized explants as targets for transformation circumvent time-consuming tissue culture steps but increases the likelihood that chimeric plants are produced.
Targeting gametes, zygotes or early stage embryos in embryo sacs for transformation is a potential solution to these problems. Transformed plants can be rapidly recovered thereby eliminating the need for a prolonged tissue culture step. The method thereby circumvents classical somatic tissue culture. Instead, the method permits recovery of transformed plants through normal embryogenesis. In addition, transformed gametes and zygotes, as well as a high percentage of early stage embryos, will give rise to uniformly transformed plants capable of transmitting the foreign gene to progeny.
A need therefore exists for an efficient method for production of transgenic plants uniformly transformed with a foreign gene. A need also exists for an efficient method for production of transformed plants capable of transmitting a foreign gene to progeny. A further need exists for a method of isolating embryo sacs that permits zygote development into an embryo which will develop into a normal whole plant. In addition, a need exists for a method for the transformation of gamete cells or zygotes.